fit-for-purpose biomarker validation of non-lba … · case study 1 –cancer vaccine ihc &...

FIT-FOR-PURPOSE BIOMARKER VALIDATION OF NON-LBA ASSAYS AND NEW TECHNOLOGIES

STEPHANIE TRAUB

EBF 11TH OPEN MEETING

22ND NOVEMBER 2018

SPAIN

DISCLAIMER

The views expressed in the presentation are those of thespeaker solely. The speaker does not make any warrantyof any kind, expressed or implied, and takes noresponsibility for the completeness, accuracy, reliability orsuitability of work based on views expressed in thepresentation for any regulatory claims.

AGENDA

1) Biomarkers – why and how?

2) Case study 1 – Cancer vaccine (IHC & ELISPOT)

3) Case study 2 – CAR-T trial (Q-PCR & Flow Cytometry)

BIOMARKERS – WHY AND HOW?

Adapted fromhttps://www.nature.com/nrc/posters/microenvironment/micro_poster.pdf

Cancer and other indications are multifactorial diseases and there is a need to measure pharmacodynamic- and immune-endpoints and patient selection Biomarkers for early decision making

Small molecules

18

Combination1

Biologicals 6


PORTFOLIO SNAPSHOT

2012

Biologicals10

Small molecules

11OCT 2018

Increase in targeted and immuno-oncology therapies


TISSUE BASED BIOMARKER ASSAYS/TECHNOLOGIES

Multi-colour IHC

Imaging Mass Cytometry

Nanostring(gene/protein signatures)

Flow Cytometry

RNA Seq

TCR Seq

Single-stain IHC

Q-PCR

Proteomics

Transcriptomics

Whole exome sequencing

(TMB)

RNAScope

ctDNA/CTCs

Mass Cytometry

ELISA

ELISPOT

Flow Cytometry

Genomics

Image StreamWES

Metabolomics

Q- PCR

Proteomics

Nanostring

miRNA

Transcriptomics

Western Blot


LIQUID BASED BIOMARKER ASSAYS/TECHNOLOGIES

MHC profiling

4654

NO. OF PROJECTS USING FLOW CYTOMETRY IN % (n=21)

yes no

18

82

9

9

9

target engagement

2

immune response

monitoring9

quantitative immune profiling

1

pentamer 1

receptor occupancy

1

“PK”1


ASSAYS BASED ON ONE TECHNOLOGY

TYPE OF FLOW CYTOMETRY ASSAY USED (n=11)

IS THE ASSAY FIT-FOR-PURPOSE?

➢ What role will the Biomarker play in the context of the trial?

• Patient selection marker?

• Primary, secondary, tertiary or research endpoint?

➢ Is the assay’s analytical performance acceptable for the context in which the Biomarker will be used? Statistical power?

➢ Can the assay measure what it is intended to measure on the types of specimens available?

Timmerman et al., Bioanalysis (2012) 4 (15), 1883-1894

Chau et al., Clin Cancer Res. 2008 Oct 1; 14(19): 5967–5976.



Adapted from Cummings et al., Br J Cancer. 2010 Oct 26; 103(9): 1313–1317.

IHC

Patient Selection

POSSIBLE BIOMARKER STRATEGY

Question

Constraints:Money/Time

Endpoint/Phase of Clinical DevelopmentGuideline/guidance available or not

Number of Assays/Technologies available:Reliability

Accessibility

Validation Strategy:Number of Validation Parameters possible/necessary

CASE STUDY 1 – CANCER VACCINE IHC & ELISPOT

tumourTumour type

% NY-ESO-1 protein expression 1

Based on E978Based on

other antibodiesNSCLC 16% 15.5%Oesophageal 11% 20%Melanoma 28% 39%Bladder/TCC 10% 19.5%Gastric adenocarcinoma 10% 13%Epithelial ovarian cancer 9% 21%Synovial sarcoma 64% 73%Chondrosarcoma 21% -Myxoid liposarcoma 88% -Testicular 12% 30%

1 based on average numbers from several publications – publications not cited

NY-ESO-1

https://media.biocompare.com/m/37/product/7013544-400x300.jpg

VALIDATION PARAMETERS – PATIENT SELECTIONIHC comments

Analytical Range ✓ Cell lines with different expression levels

Interference (Matrix or Drug) ✓ Drug: competitive assay with alternative method

Precision ✓ Cell lines; positive tissue consecutive sections

Accuracy ✓ Cell lines

Quality Controls (QCs) ✓ Embedded cell lines; known positive tissue

Selectivity ✓ Staining of known negative tissue

Specificity ✓ Alternative method: WES, Western, Flow

Sensitivity/LOD/LLOQ ✓ Cell lines with different expression levels

Carry Over

Hook Effect

Linearity of Dilution

Parallelism

Analyte Stability ✓

Sample Collection Integrity ✓ Same sample processing protocol

Inter-lab ✓

Other ✓ Antigen retrieval method

ParameterAssay

Jensen et al., Mod Pathol. 2017 Feb;30(2):180-193.https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm285297.pdf

https://www.ncbi.nlm.nih.gov/pubmed/27767098

IHC comments

Analytical Range ✓ Cell lines with different expression levels

Interference (Matrix or Drug) ✓ Drug: competitive assay with alternative method

Precision ✓ Cell lines; positive tissue consecutive sections

Accuracy ✓ Cell lines

Quality Controls (QCs) ✓ Embedded cell lines; known positive tissue

Selectivity ✓ Staining of known negative tissue

Specificity ✓ Alternative method: WES, Western, Flow

Sensitivity/LOD/LLOQ ✓ Cell lines with different expression levels

Carry Over

Hook Effect


Parallelism


Sample Collection Integrity ✓ Same processing protocol

Inter-lab ✓

Other ✓ Antigen retrieval method

ParameterAssay

Jensen et al., Mod Pathol. 2017 Feb;30(2):180-193.https://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm285297.pdf

VALIDATION PARAMETERS – TERTIARY ENDPOINT

https://www.ncbi.nlm.nih.gov/pubmed/27767098

CASE STUDY 1 – CANCER VACCINE IHC & ELISPOT

CD4 T cells

CD8 T cells

https://www.rndsystems.com/products/elispot-assay-principle

IFN-γ ELISPOT

VALIDATION PARAMETERS – SECONDARY ENDPOINTELISPOT comments

Analytical Range ✓ Cells need to be titrated for spot count

Interference (Matrix or Drug) Isolated PBMCs

Precision ✓ SEB, CEF and others

Accuracy ✓ SEB, CEF and others; PBMCs; Expanded T

Quality Controls (QCs) ✓ Stim. (> min spot count); PBMCs; Expanded T

Selectivity ✓ Mock stimulated wells

Specificity ✓ Response to irrelevant peptides

Sensitivity/LOD/LLOQ ✓ plus see other

Carry Over

Hook Effect


Parallelism

Analyte Stability ✓ PBMC viability

Sample Collection Integrity ✓ PBMC viability

Inter-lab ? Each lab own cut point?

Other ✓ Cut point determination: false positive rate

ParameterAssay

Xu et al., J Transl Med. 2008 Oct 22;6:61. doi: 10.1186/1479-5876-6-61.

ELISPOT comments

Analytical Range ✓ Cells need to be titrated for spot count

Interference (Matrix or Drug) Isolated PBMCs

Precision ✓ SEB, CEF and others

Accuracy ✓ SEB, CEF and others; PBMCs; expanded T

Quality Controls (QCs) ✓ Stim. (> min spot count); PBMCs; expanded T

Selectivity ✓ Mock stimulated wells

Specificity ✓ Response to irrelevant peptides

Sensitivity/LOD/LLOQ ✓ plus see other

Carry Over

Hook Effect


Parallelism

Analyte Stability ✓ PBMC viability

Sample Collection Integrity ✓ PBMC viability

Inter-lab ? Each lab own cut point?

Other ✓ Cut point determination: false positive rate

ParameterAssay

Xu et al., J Transl Med. 2008 Oct 22;6:61. doi: 10.1186/1479-5876-6-61.


??

CASE STUDY 2 – CAR-T Q-PCR & FLOW CYTOMETRY

T cell CAR-T

CAR-T

tumour

Plasmid

CASE STUDY 2 – CAR-T Q-PCR & FLOW CYTOMETRY

Flow Cytometry

Q-PCR

http://www.genechron.com/images/immagini_secondarie/amplification.png

https://images.novusbio.com/images2/CD34_FAB7227P_Flow_Cytometry_17828.jpg

CAR-T

Plasmid

Q-PCR comments

Analytical Range ✓ Std curve with Plasmid; Genomic input; Efficacy

Interference (Matrix or Drug) Isolated but sufficient purity

Precision ✓

Accuracy ✓

Quality Controls (QCs) ✓

Selectivity ✓ Genomic DNA; NTC; BLAST

Specificity ✓ Product sequencing; Primer dimer; Cycle no.

Sensitivity/LOD/LLOQ ✓

Carry Over

Hook Effect

Linearity of Dilution ✓

Parallelism


Sample Collection Integrity ✓

Inter-lab ✓

Other ✓ Amplicon length

ParameterAssay

Bustin et al., Clin Chem. 2009 Apr;55(4):611-22. doi: 10.1373/clinchem.2008.112797.

VALIDATION PARAMETERS – SECONDARY ENDPOINT

Q-PCR comments

Analytical Range ✓ Std curve with Plasmid; Genomic input; Efficacy

Interference (Matrix or Drug) Isolated but sufficient purity

Precision ✓

Accuracy ✓


Selectivity ✓ Genomic DNA; NTC; BLAST

Specificity ✓ Product sequencing; Primer dimer; Cycle no.


Carry Over

Hook Effect


Parallelism



Inter-lab ✓

Other ✓ Amplicon length

ParameterAssay

Bustin et al., Clin Chem. 2009 Apr;55(4):611-22. doi: 10.1373/clinchem.2008.112797.


Flow comments

Analytical Range ✓ Here yes, normally not?

Interference (Matrix or Drug) It is the drug

Precision ✓

Accuracy


Selectivity

Specificity ✓ FMI/isotype control


Carry Over ✓

Hook Effect


Parallelism



Inter-lab ✓ Assuming same model and brand

Other ✓ Antibody titration

ParameterAssay

Der Strate, EBF; ISAC; FDA 2013; Davies; Barnet; Green.

VALIDATION PARAMETERS – SECONDARY ENDPOINT

Flow comments

Analytical Range ✓ Here yes, normally not

Interference (Matrix or Drug) It is the drug

Precision ✓

Accuracy


Selectivity

Specificity ✓ FMI/isotype control


Carry Over ✓

Hook Effect


Parallelism



Inter-lab ✓ Assuming same model and brand

Other ✓ Antibody titration

ParameterAssay


Der Strate, EBF; ISAC; FDA 2013; Davies; Barnet; Green.

IHC ELISPOT Q-PCR Flow

Analytical Range ✓ ✓ ✓ ✓

Interference (Matrix or Drug) ✓

Precision ✓ ✓ ✓ ✓

Accuracy ✓ ✓ ✓

Quality Controls (QCs) ✓ ✓ ✓ ✓

Selectivity ✓ ✓ ✓

Specificity ✓ ✓ ✓ ✓

Sensitivity/LOD/LLOQ ✓ ✓ ✓ ✓

Carry Over ✓

Hook Effect


Parallelism

Analyte Stability ✓ ✓ ✓ ✓

Sample Collection Integrity ✓ ✓ ✓ ✓

Inter-lab ✓ ? ✓ ✓

Other ✓ ✓ ✓ ✓

ParameterAssay

VALIDATION PARAMETERS – FOR SPECIFIC EXAMPLES GIVEN

SUMMARY

1) Understanding the assay and the technology

➢ It’s particularities ➢ Problems and limitations ➢ Confidence/reliability (ELISPOT)➢ Consider other non-LBA parameter

2) No. of validation parameter possible/necessary?

POSSIBLE STRATEGY

THANK YOU!

cruk.org/cdd

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fit-for-purpose biomarker validation of non-lba … · case study 1 –cancer vaccine ihc &...

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